This invention relates to methods of isolating and analyzing oligosaccharides from glycoproteins. More specifically, this invention relates to laboratory methods that can be used in order to isolate derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide and O-linked glycan amino acids generally. O-linked oligosaccharides are oligosaccharides that are covalently attached to serine, threonine, or very rarely tyrosine residues on a glycoprotein.
Cancer is a disease that has a major impact on societies across the globe. According to the National Cancer Institute (2015), an estimated 1,658,370 new cases of cancer will be diagnosed and 589,430 people will die from the disease this year in the United States alone. As a result, researchers and their institutions are in a continuous search for substances that are effective in promoting anti-cancer biological activity—whether the substances prevent the onset of cancer or, alternatively, slow down or stop the growth of cancer.
Recent scientific developments by Guo et al., Curr. Opin. Chem Biol. (2009) 13 5-6, and by Lindhorst, Bielstein J. Org. Chem. (2012) 8 804-818, have shown the utility of O-linked glycan amino acids such as those linked to serine, threonine, and very rarely tyrosine as potential cancer vaccines and synthetic glycopeptides for use in general immunostimulants. Thus, a principal objective of the present invention is to provide a method for isolating O-linked oligosaccharides in order to determine the oligosaccharide anti-cancer potential.
Despite advances in the art, problems still remain. Many obstacles arise when isolating the oligosaccharides from glycoproteins that in turn limit the understanding of oligosaccharide anti-cancer activity. In particular, problems exist with isolating O-linked glycan amino acids with specificity. Current methods require the use of reductive methods that are both costly and stifle the depth of research because these methods and systems are not as sensitive or stable. For instance, in U.S. Patent Publication 2007/0105179 A1 discloses separating and detecting N- and O-linked oligosaccharides in glycoproteins via non-degradative enzymatic cleavage. This method does not provide for the identification and isolation of particular O-linked glycan amino acids—such as derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide and for general O-linked glycan amino acids—in glycoproteins that are isolated using the method disclosed herein. Therefore, a need in the art exists to address these deficiencies.
Thus, a primary object of the invention is to provide a method and system that improves upon the state of the art.
Another object of the invention is to provide a method that is simple and maximizes a preferred pH stability required to specifically isolate an O-linked glycan amino acid.
These and other objects, features, or advantages of the present invention will become apparent from the specification, drawings, and claims.